Pharmaceutical preparation and a method for the treatment of diabetes

ABSTRACT

A method of treatment of humans suffering from diabetes which involves transplantation into the human of viable porcine islets capable of producing insulin within its host. The invention also includes the particular preparation for such transplantation and its method of preparation which involves extraction from a piglet at near full term gestation (whether delivered prematurely or not) and treatment of the tissue with nicotinamide and/or any compound exhibiting similar growth promoting and cytoprotective effects and the subsequent administration of such a compound or compounds to the patient after the transplantation.

This is a Divisional of application Ser. No. 08/665,357 filed Jun. 17,1996. which in turn is a Continuation of application Ser. No. 08/385,362filed Feb. 7, 1995, now abandoned; which in turn is a Continuation ofapplication Ser. No. 08/223,945 filed Apr. 6, 1994, now abandoned.

The present invention relates to improvements in and/or relating to thetreatment of diabetes.

The present invention relates to a method of treatment of a mammalianpatient suffering from diabetes (including humans) which involves thetransplantation into the mammal of viable porcine islets capable ofproducing insulin within its host. Such transplantation to date has notbeen sufficiently successful without side effects associated withconcomitant continuous immunosuppression.

The present invention also consists in preparations useful for such amethod of treatment.

In a first aspect the present invention may broadly be said to consistin a method of treating a mammalian patient suffering from diabeteswhich comprises transplanting into the mammal an effective amount ofislets capable of producing insulin from a pig, said islets having beenextracted from a piglet at near full term gestation (whether deliveredprematurely or not) and which tissue has been treated during preparativeprocedures with nicotinamide and/or any compound exhibiting similargrowth promoting and cytoprotective effects, and said patient at leastfor a period after such transplantation is administered (preferablyorally) with nicotinamide and/or a compound exhibiting similar effects.

Preferably the administration of nicotinamide and/or a compoundexhibiting analogous effects is administered to the mammal along with asource of protein that substitutes for bovine protein including casein.

Preferably said piglet from which the islets have been extracted isnewborn.

Preferably the preparation is substantially as hereinafter described andmay include a cryogenic storage period prior to thawing andtransplantation.

In a further aspect the present invention consists in a preparationcapable of being injected into a mammalian patient to providetransplantation of a type referred to in the method of the presentinvention, said preparation having a viable insulin producing quantityof islets that have been extracted from a newborn piglet intonicotinamide and/or a compound exhibiting analogous effects.

In a further aspect the present invention consists in the saidpreparation in a cryogenically stored form.

In still a further aspect the present invention consists in atransplantable quantity of a preparation in accordance with the presentinvention having at least 100,000 porcine islets that have been and/orare in a nicotinamide containing environment and which ontransplantation are able to multiply.

This invention has established that purified newborn piglet islets,treated with nicotinamide during the preparative procedures, can besuccessfully transplanted into spontaneously diabetic mice treated withnicotinamide and a cow protein free diet (Table 1).

Without the special preparation of donor tissue, and treatment of therecipient, such transplantation is unsuccessful. Such transplantation isalso unsuccessful in normal mice which have been rendered diabetic bythe injection of a drug (streptozotocin) which poisons the insulinproducing cells.

We have successfully carried out piglet islet transplants into micewhich are born without a functional immune system and have been rendereddiabetic with the drug. These immunodeficient mice did not develop anyinfections, confirming the sterility of the islet preparations.

From these experiments the following conclusion can be drawn:xenotransplantation of islets (piglet to mouse) can be successfullycarried out under the following conditions.

(i) islets are purified under aseptic conditions, in the presence ofnicotinamide, and can be shown to produce insulin in response toglucose, before and after cryopreservation. The amount needed forsuccessful transplantation in mice is about 100-200,000 islet cells.

(ii) the recipient mouse is--

(a) either spontaneously diabetic (NOD strain) or lacks a functionalimmune system.

(b) receives both nicotinamide from at least the time transplantationand preferably also a cow protein free diet from at least the time oftransplantation.

Variation from these conditions usually (if not always) result infailure.

Preparation of newborn piglet islets

A litter of piglets are delivered by Caesarian section and theirpancreases removed under sterile surgical conditions. The pancreases arediced, and incubated with collagenase under sterile conditions. Theislets are then partially purified on a density gradient, and thenexplanted into tissue culture containing 10 m molar nicotinamide, for 1week. At the end of this time, further purification has occurred. Theislet cell are then checked for viability (dye inclusion) and ability tomake insulin in vitro, in response to glucose. The cells and culturemedium are checked for a battery of human and pig pathogens thencryopreserved. A small batch is thawed, rechecked for viability, insulinproduction in vitro, sterility, and in vivo ability to reverse diabetesand in vivo sterility.

This procedure ensured that islets stored in liquid nitrogen will beviable, and sterile when thawed prior to transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparison of the average blood glucose level and insulindosage before and after transplantation of the islets.

FIG. 2 shows a plot of the reduction in daily insulin dose relative tothe pre-transplant level against the days after transplantation.

HISTORICAL REVIEW

It has been about 100 years since the first attempt to transplant viableinsulin producing tissue--in this case from a dog, into diabetic humans.Islet cell allotransplants were first attempted in the late 1970s, andefforts continue to be made up to this time. Islets from mid-trimesterfoetuses have been uniformly unsuccessful. Only in the 1980s did theprogress of two decades of basic scientific research result in a bettertechnique to purify a high yield of human islets.

Using closely H.L.A. matched adult donors, with great care to ensureviability, purity and adequate numbers of islets (>200,000) some limitedsuccess has been attained in reversing diabetes (about 30% success at 1year of follow up. In all of these attempts, the recipient has receivedcontinuous immunosuppression (which has itself presented unwanteddangers to the recipient) usually including cyclosporine. More recently,close attention to diabetes control following transplantation has beenadded to the protocols.

Allo-transplantation of islets into diabetic subjects has, in additionto the usual problems of vascularization and rejection common to anysuch transplant, the additional problem of recurrence of `insulitis` andβ cell destruction inherent to diabetes--as exemplified by the shortterm success, but longer term failure of pancreatic segmentaltransplants between identical twins discordant for diabetes. (SeeSUTHERLAND D E R MATAS A J, GOETZ F C, NAJARIAN J S.

Transplantation of dispersed pancreatic islet tissue in Izunians:autografts and allografts. Diabetes 1980; 29 (Suppl.1): 31-44).

Whilst the ideal transplant donor tissue should be H.L.A. identical withthe recipient, this is the combination most likely to result in diseaserecurrence in the graft.

Transplantation of mice rendered diabetic with streptozotocin with donormouse islets different in several major H.L.A. loci has been carried outsuccessfully, using purification technique to eliminate non-islet cellcontaminants. (See BOWEN K M, ANDRUS L, LAFFERTY K Successfulallotransplantation of mouse pancreatic islets to non-immunosuppressedrecipients. Diabetes 1980; 29:98).

These transplants were carried out without any form of recipientimmunosuppression.

However, when similar transplants were made into spontaneously diabetic(NOD) mice, they were unsuccessful, unless the recipients were treatedwith nicotinamide and desferrioxamine, (See NAMIKOS I N, PRO WSE S J,CAROTENUTO P, LAFFERTY K J. Combined treatment with iticotinamide anddesferrioxamine prevents islet allograft destruction in NOD MICE.DL4BETES 1986; 35: 1302) which appeared to prevent disease recurrence inthe transplanted tissue. Nicotinamide can prevent diabetes in thisstrain of mouse (See YAMADA K, NONAKA K, HANAFUSA T, MIYAZAKIA,TOYOSHIMA H, TARUI S.

Preventative and therapeutic effects of large dose nicotinamideinjections on diabetes associated wilt insulitis, Diabetes 1982; 31:749-753) although its precise mode of action is subject to much debate.Desferrioxamine is thought to act as repressor of free-radicalgeneration.

Xenotransplantation (pig to streptozotocin induced diabetic mouse) ofislets has previously only been successfully carried out in athymic nudemice (See KORSGREN 0, JANSSON L, EIZINK D, ANDERSONA. Functional andmorphological differentiation of fetal porcine islet like cell clustersafter transplantation into nude mice. Diabetologia Vol 34: 379-386,1991.)

These mice lack T cells, but are able to generate antibodies to certainantigens via nonthymic dependent B cells. Whilst xenotransplantation maybe the best option to prevent disease recurrence grafts made intospontaneously diabetic animals or humans, because of greatdissimilarities in tissue antigens, the likelihood of rejection iscorrespondingly increased.

Pig islets have been prepared and injected into the portal vein of adiabetic human subject--with only transient evidence of production ofpig (pro)insulin (See KORSGREN O, GROTH C G, ANDERSON A, HELLERSTRON C,TIBELL A, TOLLEMAR J, BOLINDER J, OSTMAN J, KUMAGAI M, MOLLER E,BJOERSDORFF A. Transplantation or Porcine fetal pancreas to a DiabeticPatient. Transplantation Proceedings vol 24, No. 1 (February), 1992:352-353) xenotransplantation of other organs (spleen, liver has beennotably unsuccessful despite treatment with `state of the art`immunosuppressants (e.g. the recent Pittsburgh experiences). On theother hand, these organs cannot be rid of `lymphocytes and other activeantigen presenting cells

We have succeeded with allotransplatation into diabetic NOD mice usingneonatal donor tissue islets from the pig.

A novel feature has been the use of neonatal donor tissue as

i) islets are easier to prepare in partially purified form from veryyoung animals than older animals

ii) the islets are still capable of some replication, compared withadult islets, and the use of nicotinamide in the culture media used inislet preparation.

Islets flourish in media enriched with 1Om2 nicotinamide. Cell numbers,D.N.A content and insulin production capacity are enhanced. (See SANDLERS, ANDERSON A. Long term effects of exposure of pancreatic islets tonicotinamide in vitro on DNA synthesis, metabolism and Beta cellfunction. Diabetologia 1986; 29-199).

The replication and maturation of foetal islets is improved by suchtreatment (See SANDLER S, ANDERSON A. Stimulation of cell replication intransplanted pancreatic islets by nicotinamide treatment.Transplantation 1988; 46(1):30-31).

Cytokines which induce MHC proteins also have β cell cytotoxic effectswhich are prevented by nicotinamide (See MANDRUP-POULSEN T, BENDTZENKNIELSENJ, BENDIXEN G, NERUPJ. Cytokines cause functional and structuraldamage to isolated islets of Langerhans. Allergy 1985, 40: 424-429 andKOLB H, BURKART U, APPELS B, HANNENBERG, KANTWERK-FUNKE G, KIESEL U,FUNDA J, SCHRAERMEYEN U, KOLB-BACHOFEN V (1990). Essential contributionof macrophages to islet cell destruction in vivo and in vitro. JAutoimmun 3: 1-4).

Nicotinamide pretreatment suppresses Class 2 M.H.C. expression on βcells. (See YAMADA K, MIYAJIMA E, NONAKA KYOHEI. Inhibition ofcytokine-induced MHC class II but not class I. Molecule expression onmouse islet cells by Nicotinamide and 3 Aminobenzamide. Diabetes vol.39: September 1990: 1125-1130).

Without being tied to a theory we believe nicotinamide may thereforeprevent antigen presentation by β cells during the traumatic process ofpurification of islets from other pancreatic components, as well asproducing more and more biologically active β cells. We believe alsothat other compounds may exhibit a similar activity provided any suchcompound has functional homology with nicotinamide.

Nicotinamide alone can prevent diabetes in this strain if given earlyenough, (See REDDY S, BIBBY N, ELLIOTT R. Dietary prevention andenhancement of diabetes in the NOD mouse. Lessons from Animal DiabetesII, Third Intemational Workshop, March 1990: p 34). as can an`elemental` or cow protein free diet (See ELLIOTT R, REDDY S, BIBBY N,KIDA K. Dietary prevention of diabetes in the non-obese diabetic mouse.Diabetologia 1988, 31: 62-64).

Neither procedure alone is sufficient to prevent disease if given nearto the time when diabetes usually occurs, but given together iseffective (See BIBBY N, ELLIOTT R B. Prevention of Diabetes in the NODmouse with nicotinamide and Prosobee--Dosage and Timing are important.Abstract S. 60. Diabetes Research and Clinical Practice. Vol 14. Suppl1, 1991).

We have found that in addition to continuance of the putative effects ofnicotinamide used during the preparative procedures, an additional`antidiabetic` effect can be obtained. This can be further enhanced bythe elemental or soy protein diet.

The effects of these procedures on allotransplantation into NOD mice ofBALBC islets (given I.P.) is shown in Table 1.

    ______________________________________                                        ISLET CELL ALLOTRANSPLANTS IN                                                 NON-OBESE DIABETIC MICE (NOD)                                                                    Permanent Temporary                                                                             Total no.                                Diabetic NOD                                                                           # NOD     remission remission                                                                             remission                                mice     transplanted                                                                            #      %    #    %    #    %                               ______________________________________                                        Transplant                                                                             20        3      15   2    10   5    25                              Only                                                                          Transplant/                                                                            20        6      30   2    10   8    40                              Nicotinamide                                                                  Nicotinamide                                                                           20        1       5   4    40   5    25                              Only                                                                          ______________________________________                                    

Fisher exact Probability Test was used and the difference between thethree groups was statistically significant (0.0415).

The analysis of survival based on a proportional hazard model (PHREG)was used to assess whether or not post transplant survival was longerthan expected with the general conclusion of a statistically significantbenefit of transplantation compared with the survival experience of nontransplanted mice (Chi square 0.0009)

The survival analysis of the combined treatment compared with controlgroup shows a statistically significant benefit of this treatment (Chisquare 0.0394).

We have shown 30% permanent cure of diabetes using newborn islet cellswhich have been cultured for 7 days with Nicotinamide, cryopreserved andtransplanted (I.P.) into NOD mice given nicotinamide in drinking water.Neonatal piglet islets were similarly prepared and injected intodiabetic NOD mice (Table 2).

                  TABLE 2                                                         ______________________________________                                        PORCINE ISLET CELL ZENOTRANSPLANTS                                            IN NON-OBESE DIABETIC (NOD) MICE.                                                                Permanent Temporary                                                                             Total no.                                Diabetic NOD                                                                           # NOD Mice                                                                              remission remission                                                                             remission                                mice     transplanted                                                                            #      %    #    %    #    %                               ______________________________________                                        Casein free diet                                                                       20        2      10   3    15    5   25                              only                                                                          Nicotinamide +                                                                         20        7      35   6    30   13   65                              casein free                                                                   diet                                                                          Nicotinamide                                                                           16        1      6.3  2    12.5 13   18.8                            Transplantation                                                                        13        2      15.3 4    30.8  6   46.2                            Only                                                                          ______________________________________                                    

The effect of the treatments on the diabetic status was investigatedusing a logistic regression model Mice who had transplants wereclassified as having no remission, temporary remission or permanentremission using the number of aglycosuric days after treatment.

There was a significant difference between the treatments (p=0.0023) thenicotiamide status was found to be statistically significant (p=0.0076).

60% of the mice receiving nicotinamide and casein free diet reverseddiabetes (12/20), six of them permanently, with a significant benefitfrom this trial compared with the other three groups.

In summary, allo and xeno transplantation of islets into diabetic NODmice can be successfully carried out under the conditions of the presentinvention.

We have successfully replicated the xenotransplantation of athymic nudemice, (See KORSGREN 0, JANSSON L, EIZIRIK D AND ANDERSON A. Functionalmorphological differentiation of fetal porcine islet like cell clustersafter transplantation into nude mice. Diabetologia (1991) 34: 379-386)but have been unsuccessful with the procedure in another immunodeficient strain (severe combined immunodeficient-SCID mice). These micelack effective T & B cells, but do have active natural killer (NK)cells.

We have also been less successful in attempts to xenotransplant Swissmice (the

non-diabetic progenitors of the NOD mouse) rendered diabetic withstreptozotocin,

using the same procedures which were successful in the NOD (Table 3).

    ______________________________________                                        PORCINE ISLET CELL TRANSPLANTATION INTO                                       DIABETIC SWISS MICE (ST2) 1992                                                               Permanent Temporary Total                                             # Swiss Remission Remission Remission                                         Transplanted                                                                          #         #         #                                          ______________________________________                                        Nicotinamide +                                                                         9         1         3       4                                        Casein Free                                                                   Diet                                                                          Transplant                                                                             6         1         1       2                                        Only                                                                          ______________________________________                                    

It appears that the NOD mouse behaves immunologically more like the nudemouse, than the SCID or Swiss mouse. Lazarus et al. have demonstrated`thymic anergy` in the NOD mouse over the age of 7 weeks. (See ZIPRIS D,LABARUS A, CROW A, HADZIJA M, DELOVITCH T. Defective thymic T cellactivation by concanavalin A and Anti CD3 in autoimmune non-obesediabetic mice. The Journal of Immunology Vol. 146:3763-3771). This`anergy` results in T-cells not being effectively `trained` in thethymus, and thymic lymphocytes being unresponsive to Con A (ConcanavalinA) and anti CD3. This defect is due to a genetically determinedthymus-dependent phenomenon expressed in NOD mice.

Some credence can be given to the idea that the diabetic NOD mouse maybe partially immunodeficient.

Diabetes in the human is similar to the disease in the NOD mouse, andmay respond to similar xenotransplantation procedures. The similaritiesand dissimilarities are listed below.

                  TABLE 4                                                         ______________________________________                                                       NOD MOUSE                                                                              HUMAN                                                 ______________________________________                                        Insulin and age dependent                                                                      +          +                                                 Female > male    ++         ±                                              Insulitis, β cell destruction                                                             +*         +                                                 `HLA` association                                                                              +          ++                                                Associated endocrine                                                                           +          +                                                 immuno pathology                                                              MHC Class 2 non-aspartate                                                                      +          +                                                 (β chain 57) association                                                 Islet cell antibodies                                                                          +          +                                                 Insulin autoantibodies                                                                         +          +                                                 le deficiency    +          ?                                                 `Thymic anergy`  +          ?                                                 Incidence        120-300 days                                                                             1-80 years                                                         (post-pubertal)                                                                          (peripubertal                                                                 predominantly)                                    ______________________________________                                         *initially periinsular                                                   

The treatment of a human is as follows:

Full blood count, liver function tests, blood urea, nitrogen andcreatinine will be measured. A pregnancy test will be done whererelevant.

Normal insulin treatment will be continued up to 24 hours beforetransplantation when 4 hourly short acting insulin will be prescribedaccording to blood glucose tests. The last insulin injection will begiven 4 hours before the transplant.

A cow's milk free dies commenced 1 week before transplantation andnicotinamide 1.2 g/m² /day given as slow release preparation(Enduramide®) in the 24 hours prior to transplantation. an additional 1g of soluble nicotinamide given orally immediately prior totransplantation.

2×10⁶ islet cells (prepared and purified in the presence of 10 mMnicotinamide) suspended in saline is then injected intraperitoneallyunder local anaesthetic after checking the placement of the needleintraperitoneally by x-ray with a small amount of contrast medium.

The number of cells transplanted would, at most, contain 10-20 units ofinsulin, and therefore, produce a relatively mild insulin reaction, evenif all were killed immediately and their insulin released. Hourly bloodglucose monitoring and normal means (but without casein) followingtransplantation, and omission of the insulin injections due at the timetransplantation is performed will minimise the effect in the unlikelyevent it occurs. Normal fasting adults can tolerate 10-29 units of quickacting insulin given by subcutaneous injection.

Monitoring of response

1. Insulin requirements/24 hours to maintain near euglycemia.

2. Recurrence of islet cell antibodies.

3. C-peptide measurement of 24 hour procine urinary excretion--at aboutmonthly intervals initially used as an index of the transplant function.

Further testing (oral glucose tolerance) is conducted if insulinrequirements disappear.

The nicotinamide and C.M. protein free diet will be continued for atleast 3 months, and probably indefinitely if insulin requirementsdisappear.

Uncontrolled insulin production

It is conceivable that successfully transplanted piglet islets couldproduce insulin even when blood glucose levels are normal. This has nothappened in the piglet to mice experiments, nor in human to human(allograft) experiences internationally.

Piglets islets are killed by the drug streptozotocin, whereas humanislets are not. This drug has been used in humans to controlinappropriate insulin secretion from malignant islets which aresensitive to the drug.

The likelihood of the above complication is exceedingly remote.

CLINICAL EXAMPLES

Two such xenotranplants have been carried out in diabetic humansubjects.

The first was a 15 year old female who had diabetes for 7 yearsrequiring the injection of daily doses of insulin totalling 76-78units/day. Despite this her diabetic blood flucose levels were poorlycontrolled. The Xenotransplant was carried out as above, using 200,000islets. There was an immediate reduction in insulin requirement whichreached its maximum between the 16-21st. day post operatively. Duringthis period average blood glucose control was better thanpreoperatively. This reduction averaged 18% less than the pretanplantdose during this period. The effect slowly waned over the next fewweeks.

The second transplant involved a 15 year old diabetic male who had thedisease for 7 years. On this occasion 800,000 viable islets of more than150 u in diameter were transplanted. On this occasion the insulin doeswas reduced to a minimum of 55% of the pretransplant does in the thirdweek post transplant and averages 62% of the pretransplant does in thefifth week after transplantation. The average blood glucose levelsbefore transplantation of about 10 mm/1 have been reduced to 6.5 mm/1 inthe 4th and 5th weeks. The time course of blood glucose and insulin dosein this subject are shown in FIG. 1.

It appears that the transplanted piglet islets are capable of producinginsulin for at least 5 weeks after engraftment in diabetic humans andthat the magnitude of the effect is related to the number of isletsimplanted. The duration of the effect in the second instance indicatesthat acute rejection of the transplanted tissue has not occurred. Noside effects of the procedure have been encountered. Further transplantprocedures will be carried out using a larger number of islets but inother ways not varying the technique. To date the results in humans aresimilar to those described in the diabetic mice transplanted with pigletislets.

What is claimed is:
 1. A method for treatment of a mammalian patientsuffering from diabetes which comprises:(a) extracting neonatal pigletislet cells; (b) treating said islets with nicotinamide; (c) injectingso as to transplant into said mammalian patient and effective amount ofviable piglet islet cells which have been treated in accordance withstep (b), said viable islets being capable of producing insulin in ahost; (d) administering nicotinamide to said mammalian patient at leastsubsequent to transplantation; and (e) administering a casein-free dietto said mammalian patient.
 2. The method of claim 1, wherein themammalian patient is administered nicotinamide prior to transplantation.3. The method of claim 1, wherein the casein-free diet is administeredto the mammalian patient at least after transplantation.
 4. The methodof claim 1, wherein the casein-free diet is administered prior totransplantation.
 5. The method of claim 1, further comprising storingthe islets in a cryogenic form.
 6. The method of claim 1, wherein saidnicotinamide is administered to said mammalian patient orally.
 7. Themethod of claim 1, wherein the islets have been extracted from pigletswhich are newborn.
 8. The method of claim 1, wherein the transplantationis xenotransplantation.
 9. The method of claim 1, wherein nicotinamideis administered in a dosage of between 1.2-2.4 g/m² body surface. 10.The method of claim 1, wherein said method permits transplantationwithout the need for immunosuppressive treatment.
 11. A preparationcomprising:an effective amount of viable islets extracted from aneonatal piglet delivered within 4 weeks after birth, said islets beingcapable of producing insulin in a host and been treated duringpreparative procedures with nicotinamide, said islets being in aninjectable form for transplantation into a mammalian patient.
 12. Thepreparation of claim 11 in a cryogenic form.
 13. The preparation ofclaim 11, having at least 100,000 porcine islets and which upontransplantation are able to multiply.
 14. The preparation of claim 11,wherein said preparation permits transplantation without the need forimmunosuppressive treatment.